Unless otherwise stated, all procedures are carried out in the darkroom using a red safe light.
1. Make up troughs containing 350 mL fresh developer, stop solution, and fixing solution. Allow or adjust the temperature of all solutions to reach 20°C, and place in the darkroom.
2. Remove slides from the refrigerator, and allow them to equilibrate to room temperature.
3. Place a rack of slides in the developer for exactly 2 min and 30 s, and then directly into the stop solution for 1 min and into the fixing solution for 5 min.
4. Wash in troughs of distilled water twice, each for 10 min. Slides can now be removed from the darkroom.
5. For counterstaining, place each slide in 50 mL of 0.25% (w/v) fresh filtered tolui-dine blue for exactly 1 min. Dehydrate by rapid dipping through a graded series of ethanols (30-100% ) washing twice in 100% ethanol.
6. Then clear with xylene (Histoclear cannot be used here, since it leaves an oily film on the slide) and mount with cover slips under Depex.
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